Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.

The molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates.

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease.